Methods for culturing and analyzing cells

ABSTRACT

The present invention generally relates to methods for culturing and analyzing cells using liquid bridges.

RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 12/683,882, filed Jan. 7, 2010, which is acontinuation-in-part of U.S. patent application Ser. No. 12/617,286,filed Nov. 12, 2009, which is a continuation of U.S. patent applicationSer. No. 11/366,524, filed Mar. 3, 2006, which is a continuation ofPCT/IE2004/000115 filed Sep. 6, 2004 and published in English, claimingthe priorities of U.S. patent application Ser. Nos. 60/500,344 and60/500,345, both filed on Sep. 5, 2003. The contents of each of theseapplications are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention generally relates to methods for culturing andanalyzing cells using liquid bridges.

BACKGROUND

A broad range of therapies exist that may be used to treat patientsafflicted with diseases, such as infections, cardiovascular disease, andneoplastic disease. Use of those therapies has revealed substantialdifferences in therapeutic response among individual. Any given drug maybe therapeutic in some individuals while being ineffective in others.Further, a drug may produce adverse effects in certain individualswhereas others do not experience the same adverse reaction to the drug.Recognition of differences in drug response among individual is animportant step towards optimizing therapy.

A problem with current approaches in drug therapy is that thosetherapies are designed for treatment of large patient populations asgroups, irrespective of the potential for individual based differencesin drug response. This approach is utilized due to costs and timeassociated with producing numerous cultures of an individual's cells,which is required for screening many different drugs as potential agentsfor effective disease treatment for a particular individual. Thisproblem is particularly acute in the field of cancer therapy, in whichtumors have qualities specific to individuals and individuals respondvery differently to the same chemotherapeutic agent.

There is a need for methods that can rapidly and cost effectivelyculture and analyze an individual's cells so that personalized treatmentprotocols may be developed and implemented.

SUMMARY

The present invention provides improved methods cell culturing and drugscreening. In general, the invention involves using liquid bridges toproduce droplets having at least one cell. Liquid bridges are used toproduce sample droplets that contain reaction components for rapidanalysis of small sample volumes. Liquid bridges allow the formation ofsample droplets through the interaction of immiscible fluids. Thedroplets may be dispensed into individual vessels for culturing of thecells or mixed with drugs to analyze the affect of the drug on the cell.Use of liquid bridges provides for an automated system with highthroughput for preparing cell cultures and for analyzing affect of drugson cells. Thus the invention provides significant improvements in speedand cost associated with culturing and/or analyzing cells.

In certain aspects, methods of the invention are used for highthroughput formation of droplets wrapped in an immiscible carrier fluid,in which the droplets contain at least one cell. The droplets may thenbe dispensed and the cells are either cultured in the droplets or thedroplets are burst and the cells are cultured in the vessel to whichthey are dispensed. Droplets may be generated in numerous ways, althougha preferred method is using liquid bridges. One method of generating asample droplet including at least one cell involves dipping an openended channel into the first vessel to produce a sample dropletincluding at least one cell. Another method of generating the sampledroplet involves flowing a sample fluid including the plurality of cellsto a liquid bridge, in which the liquid bridge segments the sample fluidto produce the wrapped sample droplets.

Prior to dispensing, the sample droplets may be mixed with cell culturemedia to produce a mixed wrapped droplet. Droplet mixing may beaccomplished by flowing the sample droplets to a liquid bridge to mixwith droplets of cell culture media to produce the mixed wrappeddroplet. The sample droplets and the cell culture droplets may flowthrough a same channel, and flow may be used to cause the droplets tomix at the liquid bridge to form the mixed wrapped droplet.Alternatively, the sample droplets and the cell culture media dropletsmay flow through different channels and meet for mixing at the liquidbridge.

Methods of the invention involve dispensing the sample droplets tovessels for culturing. In particular embodiments, the cells aredispensed to individual wells of a well plate, e.g., 96 well or 384 wellplate. Dispensing may be accomplished by generating a siphoning effect,and then using the siphoning affect to dispense the droplets. In certainembodiments, the droplets are dispensed without a substantial amount ofcarrier fluid.

Methods of the invention may be used to prepare any type of cell forculturing, such as mammalian cells, e.g., human cells. In certainembodiments, the cells are obtained from a human tissue or body fluid.In particular embodiments, the cells are obtained from a tumor. In otherembodiments, the cells are brain cells or embryonic cells. The startingmixture of cells may be either homogeneous (i.e., a mixture includingall the same type of cells) or heterogeneous (i.e., a mixture includinga plurality of different types of cells).

Another aspect of the invention provides methods for analyzing theaffect of at least one agent on a cell or cellular component. Thosemethods of the invention involve forming a droplet wrapped in animmiscible carrier fluid, in which the droplet includes at least onecell or cellular component and at least one agent, and analyzing thewrapped droplet to determine the affect of the agent on the cell orcellular component. Analyzing of the cells may occur while the wrappeddroplets are flowing through a channel. Alternatively, the droplets maybe dispensed to a vessel, such as individual wells of a well plate,prior to analysis. In certain embodiments, a liquid bridge is used toform the wrapped droplets. In certain embodiments, the cells are lysedprior to being analyzed.

Methods of the invention may be used to analyze affect of any agent, orcombination of agents, on any cell or combination of cells. In certainembodiments, the cell is a cancerous cell and the agent is ananti-cancer agent or a combination of anti-cancer agents. In otherembodiments, different concentrations of the same agent are analyzed.

DETAILED DESCRIPTION

The present invention generally relates to methods for culturing andanalyzing cells using liquid bridges. Certain aspects of the inventionprovide methods for culturing cells. The starting mixture of cells maybe either homogeneous (i.e., a mixture including all the same type ofcells) or heterogeneous (i.e., a mixture including a plurality ofdifferent types of cells). Methods of the invention involve generatingsample droplets wrapped in an immiscible carrier fluid including atleast one cell.

Wrapped droplet generation may be accomplished by numerous techniques.The wrapped droplets may be formed, for example, by dipping an openended tube into a vessel. Exemplary sample acquisition devices are shownin McGuire et al. (U.S. patent application Ser. No. 12/468,367), thecontents of which are incorporated by reference herein in theirentirety. Parameters such as channel diameter, dipping time, and systemflow, may be adjusted so that wrapped droplets are formed in which eachdroplet contains only a single cell.

Another method for generating wrapped droplets involves using a liquidbridge. The liquid bridge is used to segment a flow of sample fluid intoindividual wrapped droplets. The droplets formed in a liquid bridge areenveloped in an immiscible carrier fluid. A typical liquid bridge of theinvention is formed by an inlet in communication with a chamber that isfilled with a carrier fluid. The carrier fluid is immiscible with samplefluid flowing through the inlet into the chamber. The sample fluidexpands until it is large enough to span a gap between the inlet and anoutlet in communication with the chamber. Droplet formation isaccomplished by adjusting flow rate, resulting in formation of anunstable funicular bridge that subsequently ruptures from the inlet.After rupturing from the inlet, the sample droplet enters the outlet,surrounded by the carrier fluid from the chamber. Further description ofusing liquid bridges for droplet formation is shown in Davies et al.(International patent publication number WO 2007/091228), the contentsof which are incorporated by reference herein in their entirety. Becausedroplet formation is controlled by flow rate, the flow rate may beadjusted to ensure that droplets are formed in which each dropletcontains only a single cell.

In certain embodiments, droplets are formed such that each dropletcontains only a single cell. Poisson statistics dictate the dilutionrequirements needed to ensure that each wrapped droplet contains only asingle cell. For example, if, on average, each wrapped droplet is tocontain only a single cell, about 1/3 of the droplets will be empty andcontain no cell, about 1/3 will contain exactly one cell, and about 1/3will contain two or more cells.

The droplet population may be enriched to maximize the fraction thatstarted with a single cell. For example, the population of cells may befluorescently tagged and thus it is possible to flow sort the wrappeddroplets after droplet formation to enrich for those that arefluorescent rather than empty. High-speed flow sorters, such as theMoFlo (Beckman-Coulter, Inc., Fullerton, Calif.), are capable of sortingat rates in excess of 70,000 per second and can be used to enrich apopulation of wrapped droplets of the invention. Similarly, it ispossible to exploit other differences between empty and full wrappeddroplets (e.g., buoyant density) to enrich a population of droplets. Inorder to enrich for droplets with one cell as opposed to several cells,it may be desirable to skew the Poisson distribution accordingly.

In certain embodiments, the sample droplets may be mixed with cellculture media prior to dispensing of the droplet to produce a mixedwrapped droplet including at least one cell and culture media. Anydevice that is capable of mixing sample droplets to form mixed sampledroplets wrapped in an immiscible carrier fluid may be used with methodsof the invention. An exemplary droplet mixing device is a liquid bridge.For droplet mixing in a liquid bridge, the sample droplet containing thecell flows to an inlet and enters a chamber that is filled with acarrier fluid. The carrier fluid is immiscible with the sample droplet.The sample droplet expands until it is large enough to span a gapbetween inlet and outlet ports. Droplet mixing can be accomplished inmany ways, for example, by adjusting flow rate or by introducing adroplet of cell culture media to the sample droplet from a second inlet,forming an unstable funicular bridge that subsequently ruptures from theinlet. After rupturing from the inlet, the mixed sample droplet entersthe outlet, surrounded by the carrier fluid from the chamber. Furtherdescription of using liquid bridges for droplet mixing is shown inDavies et al. (International patent publication number WO 2007/091228).

The sample droplets and the cell culture media droplets may flow througha same channel, and flow may be used to cause the droplets to mix at theliquid bridge to form the mixed wrapped droplet. Alternatively, thesample droplets and the cell culture media droplets may flow throughdifferent channels and meet for mixing at the liquid bridge.

After droplet generation, the cells in the droplets are cultured. Incertain embodiments, the wrapped droplets may are dispensed to a vesselor vessels for culturing of the cells in the droplets. In particularembodiments, the vessel is a well plate, e.g. 96 well or 384 well, andthe droplets are dispensed to individual wells of the well plate.Droplet dispensing may be accomplished in numerous ways. In certainembodiments, a system is configured to produce a siphoning effect. Thesiphoning effect refers to flow that is driven by a difference inhydrostatic pressure without any need for pumping. The effect isproduced by configuring a system such that a dispensing end or port islower than a fluid surface at an acquisition point, e.g., a sampleacquisition stage. The system may include any number of additionalcomponents that are positioned at an intermediate point in the system.Those intermediate components may be higher or lower than theacquisition point as long as the dispensing end is lower than the fluidsurface at the acquisition point.

The siphoning effect drives flow through the system and is used todispense the droplets. The siphoning allows for dispensing of individualintact sample droplets, and thus individual intact cells for culturing.Further description regarding systems driven by a siphoning effect isshown in Davies et al. (U.S. patent application Ser. No. 12/683,882,filed Jan. 7, 2010, and entitled “Sample Dispensing”), the contents ofwhich are incorporated by references herein in their entirety.

In certain embodiments, the cells are cultured in the wrapped droplets.Media exchange may be accomplished by re-acquiring the droplets into theliquid bridge system using a sample acquisition device such as onedescribed in McGuire et al. (U.S. patent application Ser. No.12/468,367, filed May 19, 2009, and entitled “Sampling Device), thecontent of which is incorporated by reference herein in its entirety.

Once the droplet has been acquired, the sample droplet is flowed to aliquid bridge where it is segmented, forming two droplets: a droplet ofused culture media; and a droplet containing the cultured cells in areduced volume of culture media. Droplet segmentation is described forexample in Davies et al. (U.S. patent application number 2008/0277494),the content of which is incorporated by reference herein in itsentirety.

After segmentation, the droplets are then flowed to a subsequent liquidbridge where a droplet of fresh cell culture media is introduced to thedroplet containing the cell culture. In this manner, old culture mediais removed from the cell culture and the cell culture is provided withfresh media. The droplet containing the cell culture is subsequentlydispensed to a vessel for continued culturing or analysis and thedroplet containing solely media is dispensed to waste. This process maybe repeated as many times as necessary.

In other embodiments, the droplets may be dispensed to a vessel aswrapped droplets that are subsequently ruptured in the vessel. Incertain embodiments, it is advantageous to ensure that a substantialportion of the carrier fluid is not dispensed into the collectingvessel. In one manner, flow rate is used to ensure that a substantialportion of the carrier fluid is not dispensed into the collectingvessel. The flow is controlled such that the dispensing port can bemoved over a waste container to dispense the carrier fluid surroundingthe droplets, and then moved over a collecting vessel to dispense thesample droplets. In this manner, a substantial a portion of the carrierfluid is not dispensed into the collecting vessel. Movement of thedispensing port is controlled by at least one robotics system.

Cells can be grown in suspension or adherent cultures. Some cellsnaturally live in suspension, without being attached to a surface, suchas cells that exist in the bloodstream. There are also cell lines thathave been modified to be able to survive in suspension cultures so thatthey can be grown to a higher density than adherent conditions wouldallow. Adherent cells require a surface, such as tissue culture plasticor microcarrier, which may be coated with extracellular matrixcomponents to increase adhesion properties and provide other signalsneeded for growth and differentiation. Most cells derived from solidtissues are adherent. Depending on the type of cells to be cultured, thedroplets may contain a support matrix or microcarrier. The liquid bridgemay be used to introduce the microcarrier to the droplet containing thecells.

Once dispensed, the cells are cultured according to standard methods andprotocols known to one of skill in the art. See for example, J. W.Pollard and J. M. Walker (Basic Cell Culture Protocols, 2nd ed.: Methodsin Molecular Biology, Vol. 75, Humana Press, 1997); J. Davis (Basic CellCulture: A Practical Approach, 2nd ed., Oxford University Press, 2002);S. Ozturk and W. Hu (Cell Culture Technology for Pharmaceutical andCell-Based Therapies, CRC Press, 2005); A. Doyle and J. B. Griffiths(Cell and Tissue Culture for Medical Research, John Wiley & Sons, Ltd,2000); A. Doyle, J. B. Griffiths (Cell and Tissue Culture: LaboratoryProcedures in Biotechnology, John Wiley & Sons, Ltd, 1998); R. IanFreshney (Culture of Animal Cells: A Manual of Basic Techniques, 5thed., John Wiley & Sons, 2005); G. Vunjak-Novakovic and R. I. Freshney(Culture of Cells for Tissue Engineering, John Wiley & Sons, 2006); andR. Pfragner and R. Freshney (Culture of Human Tumor Cells, John Wiley &Sons, 2003). The contents of each of the above is incorporated byreference herein in its entirety.

Methods of the invention may be used with any cells. In certainembodiments, the cells are mammalian cells, such as human cells oranimal cells. In other embodiments, the cells are cells from a humantissue or body fluid. A tissue is a mass of connected cells and/orextracellular matrix material, e.g. skin tissue, nasal passage tissue,CNS tissue, neural tissue, eye tissue, liver tissue, kidney tissue,placental tissue, mammary gland tissue, gastrointestinal tissue,musculoskeletal tissue, genitourinary tissue, bone marrow, and the like,derived from, for example, a human or other mammal and includes theconnecting material and the liquid material in association with thecells and/or tissues. A body fluid is a liquid material derived from,for example, a human or other mammal. Such body fluids include, but arenot limited to urine, sputum, stool, mucous, saliva, blood, plasma,serum, serum derivatives, bile, phlegm, sweat, amniotic fluid, mammaryfluid, and cerebrospinal fluid (CSF), such as lumbar or ventricular CSF.The cell may also be from a fine needle aspirate or biopsied tissue. Inparticular embodiments, the cells are tumor cells. In other embodiments,the cells are brain cells or embryonic cells.

Another aspect of the invention provides methods for analyzing effect ofat least one agent on a cell or cellular component. Methods of theinvention involve forming a droplet wrapped in an immiscible carrierfluid, in which the droplet includes at least one cell or cellularcomponent and at least one agent, and analyzing the wrapped droplet todetermine effect of the agent on the cell or cellular component. Methodsof the invention may be used to analyze effect of any agent, orcombination of agents, on any cell or combination of cells. In certainembodiments, the cells are mammalian cells, such as human cells oranimal cells. In other embodiments, the cells are cells from a humantissue or body fluid. In particular embodiments, the cells are tumorcells. The starting mixture of cells may be either homogeneous (i.e., amixture including all the same type of cells) or heterogeneous (i.e., amixture including a plurality of different types of cells).

In certain embodiments, methods of the invention are used to analyzespecific cellular components, such as a nucleolus, a nucleus, aribosome, a vesicle, a rough endoplasmic reticulum, a Golgi apparatus,cytoskeleton, a smooth endoplasmic reticulum, a mitochondria, a vacuole,a cytoplasm, a lysosome, or centrioles within centrosome. In particularembodiments, the cellular component is nucleic acid, such as DNA or RNA.Accordingly, cell may be lysed by using a liquid bridge to introduce adroplet containing a lysing agent to the droplet containing the cell.Formation of the mixed droplet mixes the lysing agent with the cell,lysing the cell and releasing the internal cellular components forsubsequent analysis. The lysing agent may be a chemical agent, such assurfactants, solvents, or antibiotics.

Droplet generation may be accomplished as described above, and dropletgeneration may be controlled as described above to produce droplets thatinclude only a single cell. Alternatively, droplets may be formed thatinclude a plurality of cells.

The agent may be any agent or combination of agents. Exemplary agentsare shown in The Merck Index (14^(th) edition. Whitehouse Station, NewJersey, 2009), the contents of which are incorporated by referenceherein in their entirety. In particular embodiments, the agent is ananti-cancer agent. Exemplary anti-cancer agents are shown in The MerckIndex (14^(th) edition. Whitehouse Station, New Jersey, 2009) and Dorret al. (Cancer Chemotherapy Handbook, 2d edition, pages 15-34, Appleton& Lange, Connecticut, 1994), the contents of each of which areincorporated by reference herein in their entirety. In particularembodiments, the anti-cancer agent is altretamine, asparaginase,bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisplatin,cyclophosphamide, cytarabine, dacarbazine, actinomycin D, docetaxel,doxorubicin, imatinib, etoposide, fludarabine, fluorouracil,gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan,methotrexate, mitomycin, mitotane, mitoxantrone, paclitaxel, topotecan,vinblastine, vincristine, vinorelbine, or combination thereof.

In certain embodiments, different concentrations of the same agent isprovided to different cells, thus providing an ability to determine aneffective dosage level or a toxicity level of an agent.

Any device that is capable of forming a sample droplets wrapped in animmiscible carrier fluid may be used with methods of the invention. Anexemplary droplet device is a liquid bridge. Wrapped droplet formationusing liquid bridges is described above. The sample droplets and theagent droplets may flow through a same channel, and flow may be used tocause the droplets to mix at the liquid bridge to form the mixed wrappeddroplet. Alternatively, the sample droplets and the agent droplets mayflow through different channels and meet for mixing at the liquidbridge.

After droplet mixing, the cell or cells in the mixed wrapped droplet areanalyzed to determine the effect of the agent on the cell or cellularcomponent in the droplet. Analyzing may be continuously, periodically orafter a specific period of time. Analyzing involves monitoringcharacteristic analog outcome measures, which may include, but are notlimited to: analog size, shape, density, color or opacity; changes incell number; cell death or proliferation; changes in secreted materialssuch as cytokines, growth factors, hormones or extracellular matrixcomponents; genetic markers that are up- or down-regulated during theculture period such as genes for receptors, cytokines, integrins,extracellular matrix molecules, or enzymes; and cell-surface moleculesincluding integrins and receptors.

One method analyzing the cell or cells includes performing a cellviability and/or proliferative capacity assay. For example, Trypan blueis one of several stains recommended for use in dye exclusion proceduresfor viable cell counting. This method is based on the principle thatlive cells do not take up certain dyes, whereas dead cells do. Trypanblue is introduced to the cell or cells and the cells are observed foruptake of the stain. A hemocytometer may be used to observe the cell orcells. Methods for performing this assay are well known in the art.

Another method for analyzing the cell or cells includes RNA extractionand analysis by reverse transcriptase-polymerase chain reaction(RT-PCR). Cells in the construct are lysed and total RNA extracted withTRIZOL (Life Technologies, Rockville, Md.). Equal amounts (1 μg) oftotal RNA are subjected to reverse transcription into cDNA at 42° C. forone hour with oligo (dt₁₈) primers. The transcripts are then beamplified by RT-PCR. Sample cDNA are also amplified for housekeepinggenes such as rRNA subunit S14 for controls.

Another method for analyzing the cell or cells includescytological-immunocytochemical analysis of cell products. Cells arefixed and stained with either hematoxylin-eosin (H&E) or withmonospecific antibodies. Immunostaining for the marker or product ofinterest is visualized using immunoperoxidase technique with a substratesuch as diaminobenzidine which will yield a brownish color. Cellpreparations are counterstained with 0.5% toluidine blue.

Another method for analyzing the cell or cells includes western blots.Cells may be resuspended in lysis buffer containing protease inhibitors.Protein concentrations may be determined and total cell extracts may beelectrophoresed on 7% SDS-polyacrylamide gel overlaid with stacking gel.The proteins are transferred from gel to nitrocellulose paper toimmunolocalize the product using monospecific antibodies. To visualizethe bands, the membrane blot may be exposed to BioMax Light Film (VWR,NJ).

Another method for analyzing the cell or cells includes determination ofparticular enzyme products. Enzymatic products may be assessed byserially diluting media samples and analyzing enzymatic activity usingcommercially available kits based on an enzyme linked immunoasssay.

Other methods of analyzing the cells include ELISA assays, or microarraytechnology.

Analyzing of the cells may occur while the wrapped droplets are flowingthrough a channel. For example, for the cell viability assay, the mixedwrapped droplet may flow to a subsequent liquid bridge for an additionalmixing step in which the mixed droplet mixes with a droplet of Trypanblue. The droplet now containing the cell(s), agent, and Trypan blue isflowed to a detection device (such as a CCD camera) that images thecells in the droplets as the droplets are flowing through the channel.

Alternatively, the droplets may be dispensed to a second vessel prior toanalysis. Droplet dispensing may be accomplished as described above.Exemplary vessels include plates (e.g., 96 well or 384 well plates),eppendorf tubes, vials, beakers, flasks, centrifuge tubes, capillarytubes, cryogenic vials, bags, cups, or containers. In particularembodiments, the second vessel is a well plate and the droplets aredispensed into individual wells of the well plate, one droplet per well.

In certain embodiments, the droplet including the cell or cellularcomponent is not mixed with an agent. Rather, the unmixed droplet isdispensed to at least one secondary vessel that already includes theagent. For example, droplets are dispensed to individual wells of a wellplate in which each well of the plate already includes an agent orcombination of agent. After dispensing, the cells are analyzed to assessthe effect of the agent or combination of agents on the cell or cellularcomponent.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

1. A method for analyzing the affect of at least one agent on a cell orcellular component, the method comprising the steps of: forming adroplet wrapped in an immiscible carrier fluid, wherein the dropletcomprises at least one cell or cellular component and at least oneagent; and analyzing the wrapped droplet to determine effect of theagent on the cell or cellular component.
 2. The method according toclaim 1, wherein prior to the analyzing step, the method furthercomprises dispensing the mixed wrapped droplet to at least onecollecting vessel.
 3. The method according to claim 1, wherein prior tothe analyzing step, the method further comprises lysing the cell.
 4. Themethod according to claim 1, wherein prior to the analyzing step, themethod further comprises establishing a siphoning effect to dispense themixed wrapped droplet.
 5. The method according to claim 1, wherein aliquid bridge is used to form the wrapped droplet.
 6. The methodaccording to claim 1, wherein the agent is an anti-cancer agent.
 7. Themethod according to claim 6, wherein the cell is a cancerous cell.
 8. Amethod for analyzing effect of at least one agent on a cell or cellularcomponent, the method comprising the steps of: generating sampledroplets wrapped in an immiscible carrier fluid, wherein the dropletscomprise at least one cell or cellular component; dispensing the wrappeddroplets to at least one vessel comprising at least one agent; andanalyzing the effect of the agent on the cells.
 9. The method accordingto claim 31, wherein the droplets comprise a single cell.
 10. The methodaccording to claim 8, wherein prior to the dispensing step, the methodfurther comprises lysing the cell in the wrapped droplet.
 11. The methodaccording to claim 8, wherein generating comprises: flowing a samplefluid comprising a plurality of cells to a liquid bridge, wherein theliquid bridge segments the sample fluid to produce the wrapped sampledroplets.
 12. The method according to claim 8, wherein generatingcomprises: dipping an open ended channel into a vessel to produce asample droplet comprising at least one cell.
 13. A method for culturingcells, the method comprising: generating sample droplets wrapped in animmiscible carrier fluid, wherein the droplets comprise at least onecell; and culturing the cells.
 14. The method according to claim 11,further comprising mixing the sample droplet with cell culture media.15. The method according to claim 14, wherein mixing comprises: flowingthe sample droplet to a liquid bridge to mix with a droplet of cellculture media to generate a mixed wrapped droplet.
 16. The methodaccording to claim 13, wherein the wrapped droplet further comprises asupport matrix.
 16. The method according to claim 13, wherein prior tothe culturing step, the method further comprises dispensing thedroplets.
 18. The method according to claim 13, wherein prior to theculturing step, the method further comprises establishing a siphoningeffect, wherein the siphoning effect is used to dispense the wrappeddroplets
 19. The method according to claim 13, wherein generatingcomprises: flowing a sample fluid comprising a plurality of cells to aliquid bridge, wherein the liquid bridge segments the sample fluid toproduce the wrapped sample droplets.
 20. The method according to claim13, wherein generating comprises: dipping an open ended channel into avessel to produce a sample droplet comprising at least one cell.
 21. Themethod according to claim 13, wherein the cells are obtained from ahuman tissue or body fluid.
 22. The method according to claim 13,wherein the cells are tumor cells.
 23. The method according to claim 13,wherein the cells are brain cells.
 24. The method according to claim 13,wherein the cells are embryonic cells.
 25. The method according to claim13, wherein the cells are cell aggregates.